Somatic calcium level reports integrated spiking activity of cerebellar interneurons in vitro and in vivo

Abstract

We examined the relationship between somatic Ca 2ϩ signals and spiking activity of cerebellar molecular layer interneurons (MLIs) in adult mice. Using two-photon microscopy in conjunction with cell-attached recordings in slices, we show that in tonically firing MLIs loaded with high-affinity Ca 2ϩ probes, Ca 2ϩ -dependent fluorescence transients are absent. Spike-triggered averages of fluorescence traces for MLIs spiking at low rates revealed that the fluorescence change associated with an action potential is small (1% of the basal fluorescence ] i is not a direct readout of the firing activity, but rather reflects the time integral of this activity. MATERIALS AND METHODSAll experimental procedures were designed in accordance with the animal care guidelines of our host institution, which have been approved by the "Prefecture de Police" (approval number A-750607) following inspection by Veterinary Services of the City of Paris and by representatives of the French Ministry of Research and the Ministry for Health, in agreement with European Directive 86/609/EEC regarding the protection of animals used for experimental and other scientific purposes. In Vitro Patch-Clamp Recording and ImagingSlice preparation. Sagittal cerebellar slices (200 m thick) were prepared from C57BL/6J mice and from PVϪ/Ϫ mice. The PVϪ/Ϫ animals are congenic with C57BL/6J, after 10 matings of heterozygous (PVϩ/Ϫ) mice with wild-type C57BL/6J animals (Moreno et al 2011). Mice aged 22-33 days were decapitated after cervical dislocation. Slices were cut at 4°C in a saline solution of the following composition (in mM): 87 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 ,

Spatially Selective Holographic Photoactivation and Functional Fluorescence Imaging in Freely Behaving Mice with a Fiberscope

“…In the absence of ChR2 expression, we found no change in GCaMP5-G fluorescence (mean DF/F p = 0.1%, where DF/F p is the calcium signal amplitude; see Figure 3B, bottom trace, and Supplemental Experimental Procedures; 95% confidence interval of the mean (CI95), [À0.7%, 0.9%]; p = 75 mW/mm 2 ; n = 48 cells from 3 mice). In ChR2/GCaMP5-G-coexpressing neurons, the evoked signal rose linearly with the number of pulses, similar to previous observations at MLI soma with fura-2 imaging in cerebellar slices (Franconville et al, 2011). Therefore, each pulse drove a ChR2-mediated cation influx, leading to an increase of GCaMP5-G fluorescence.…”

Measuring the Firing Rate of High-Resistance Neurons with Cell-Attached Recording

“…Because the fluorescence signal illustrated in Figure 7 is a sublinear function of firing frequency (Franconville et al, 2011) and because this signal increases by a ratio of threefold to ninefold during patching (physiological temperature data), the relative frequency increase induced by patching is if anything larger than this ratio. Thus, applying the calibration procedure of Franconville et al to the data obtained in the older group at physiological temperature, the firing frequency before patching is estimated at only 1.4 versus 7.1 Hz during patching, a fivefold ratio, compared with the 2.9-fold ratio indicated by the data of Figure 7C.…”

“…Muscimol stops cell firing and allows to measure the fluorescence level displayed without action potentials. Thus, the fluorescence change observed during muscimol application reflects the cell firing rate (Franconville et al, 2011).…”

“…Calcium imaging experiments were performed in slices loaded with fura-2 AM following procedures described previously (Franconville et al, 2011). In short, two-photon laser scanning microscopy was performed on MLIs from slices incubated for 40 min at 34°C in a saline containing 2 M fura-2 AM.…”

“…However, we have recently developed an optical method to study the firing rate of MLIs in slices that have been preloaded with the Ca 2ϩ -sensitive dye fura-2 (Franconville et al, 2011). This method does not require patch clamping and therefore allows to compare the firing rate with and without cell-attached recording.…”